Modified plants

ABSTRACT

A method for controlling endosperm size and development in plants. The method employs nucleic acid constructs encoding proteins involved in genomic imprinting, in the production of transgenic plants. The nucleic acid constructs can be used in the production of transgenic plants to affect interspecific hybridisation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/058,825, filed on Jan.30, 2002, which is a continuation of International Application No.PCT/GB00/02953, internationally filed Jul. 31, 2000, which was publishedin English, and claims priority to Great Britain Application No.9918061.4, filed Jul. 30, 1999, all of which are incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for controlling endosperm sizeand development, and seed viability in plants. The invention alsorelates to nucleic acid constructs for use in such methods, as well asmodified plants per se.

2. Related Art

Yield in crop plants where seed is the harvested product is usuallydefined as weight of seed harvested per unit area (Duvick, 1992).Consequently, individual seed weight is regarded as a major determinantof yield. Most monocotyledonous plants e.g. maize, wheat, (see Esau,1965) produce albuminous seeds—that is, at maturity they contain a smallembryo and a relatively massive endosperm. Consequently, inmonocotyledonous plants, the endosperm represents a significantcomponent of seed yield. Endosperms accumulate a store diversesubstances, including starch, proteins, oils and fats.

Therefore, in monocotyledons increasing the size of the endosperm or itsability to accumulate storage products is likely to increase individualseed weight and perhaps total yield.

Endosperms are utilized commercially in diverse ways, either indirectlyas part of the whole seed or directly following their purification, orthe purification of certain of their constituents. Hence endosperms mayrepresent either a proportion or the entire commercial value of a crop.

Examples of indirect usage include fodder maize and whole wheat flour.An example of direct usage of the complete endosperms is in theproduction of white flour for bread-making. Finally, maize oilrepresents an example of the utilization of a constituent of theendosperm, but there are many others.

In contrast to monocotyledons, most dicotyledonous plants, e.g. oil seedrape, soybean, peanut, Phaseolus vulgaris (e.g. kidney beans, whitebeans, black beans), Vicia faba (broad bean), Pisum sativum (green pea),Cicer aeietinum (chick pea), and Lens culinaris (lentil) produceexalbuminous seeds—that is, mature seeds lack an endosperm. In suchseeds the embryo is large and generally fills most of the volume of theseed, and accounts for almost the entire weight of the seed. Inexalbuminous seeds the endosperm is ephemeral in nature and reachesmaturity when the embryo is small and highly immature (usuallyheart/torpedo stage). Commonly embryo development depends on thepresence of the endosperm, which is generally accepted to act as asource of nutrition for the embryo.

Scott et al. (1998) showed that the size of the endosperm in terms ofthe number of endosperm cells at maturity in the dicotyledonous plantArabidopsis thaliana, a close relative of oil seed rape (Brassicanapus), is positively correlated with the weight of the mature seed.Plants that developed seeds with 80% smaller endosperms (average=80nuclei) compared to controls (mean of 2x-2x (diploid plant crosses) and4x-4x (tetraploid plant crosses)=400 nuclei) produced seeds that were46% smaller (in weight terms=14 μg) than the controls (mean of 2x-2x and4x-4x=30 μg). In contrast, plants that developed seeds with 160% biggerendosperms (average=640 nuclei) compared to controls (mean of 2x-2x and4x-4x=400 nuclei) produced seeds that were 180% larger (in weightterms=54 μg) than the controls (mean of 2x-2x and 4x-4x=30 μg).Arabidopsis seed in common with most other dicotyledonous seed iscomposed almost entirely of embryo. Hence the change in seed weight isalmost completely due to a change in embryo weight.

Consequently, modifying endosperm size, in terms of the number of cellsat maturity, has a dramatic impact on seed weight in seeds that do notcontain endosperm at maturity. Without being bound by the following, onereasonable hypothesis is that a larger endosperm accumulates a greaterquantity of reserves from the seed parent, perhaps by acting as astronger “sink”. These reserves then provide more resources forutilization by the growing embryo, resulting in a larger seed.Alternative mechanisms might operate, however.

The seeds of dicotyledons, like those of monocotyledons are utilized indiverse ways. For example, pulses such as soybean, peanut, Phaseolusvulgaris (e.g. kidney beans, white beans, black beans), Vicia faba(broad bean), Pisum sativum (green pea), Cicer aeietinum (chick pea),Lens culinaris (lentil) are important world crops that are used directlyfor animal and human consumption. Seeds of oil rape, sunflower andlinseed are processed to produce oils.

Clearly, despite the differences in the structure of monocot and dicotseeds, particularly with respect to the presence or absence of endospermin mature seeds, the size of the endosperm is an important factor indetermining individual seed weight, and therefore potentially total cropyield in plants where seed is the economic harvest. Indeed, Hannah andGreene (1998) showed that maize seed weight is dependent on the amountof endosperm ADP-glucose pyrophosphorylase, the enzyme responsible forsupplying substrate for starch synthesis.

However, there is some evidence that an increase in seed weight isassociated with a reduction in seed number in many breeding populations.Consequently, increasing individual seed size may not result in anincrease in total yield. While maize breeding programmes have beensuccessful and genetic improvement has played a significant role inincreased maize yields, the genetic component to yield has led to only adoubling of this parameter since the 1930s (Duvick, 1992). The increasein maize yields is currently less than 1% per year.

The genetic basis for the resistance to increased seed weightencountered in conventional breeding programmes is not understood.However, Giroux et al. (1996) showed that a single gene mutation in theendosperm specific gene shrunken2 of maize resulted in a seed weightincrease of 11-18% without a reduction in seed number. This suggeststhat yield improvements are possible in a plant with a long history ofintensive and successful breeding efforts, and may therefore begenerally achievable. Similarly, Roekel et al. (1998) showed thatintroduction of the tzs gene into Brassica napus results in asignificant increase in seed yield introduced for by increased seednumber per silique and increased seed weight.

There is also evidence that seed size (weight) is positively correlatedwith a number of components of “seed quality” such as percentgermination (Schaal, 1980: Alexander and Wulff, 1985; Guberac et al.,1998); time to emergence (Winn, 1985; Wulff, 1986); durability (survivalunder adverse growing conditions) (Krannitz et al., 1991); Manga andYadav, 1995); growth rate (Marshall, 1986) and yield (Guberac et al.,1998). Seed quality is an important factor in the cost of production ofcommercial seed lots since these must be tested before sale.Consequently, increasing total seed weight, even without increases intotal seed yield may have economic benefits through improvements in seedquality.

We have recently demonstrated (Scott et al., 1998) that hybridizingArabidopsis plants of different ploidies has reproducible and dramaticeffects on the weight of progeny seed. For example, an interploidy crossbetween a diploid (2x) seed parent and a tetraploid (4x) pollen parent(2x-4x) results in seed which is 240% larger than 2x-2x seed.Conversely, 4x-2x crosses result in a reduced seed size (60% of 2x-2x).Analysis of endosperm development in these F1 seed reveals a clearcorrelation between final seed size and the size of the endosperm. Incommon with most dicots, endosperm is not present in the matureArabidopsis seed but is required to nourish the developing embryo.Therefore, increased endosperm size translates into increased seed sizeby increasing embryo size, presumably by accumulating and then supplyingincreased nutrition, or by some other less direct means enabling theembryo to accumulate more resources from the mother.

In wild type 2x-2x crosses the endosperm is triploid and is formed bythe fertilization of a pair of fused haploid polar nuclei of maternalorigin with a haploid sperm of paternal origin. Consequently, there is a2:1 ratio of maternal to paternal genomes in the normal endosperms. Anexcess of paternal genomes in the endosperm, e.g. as a result of a 2x-4xcross, causes increased endosperm proliferation (hyperplasia). An excessof maternal genomes in the endosperm (4x-2x crosses) has the oppositeeffect: decreased endosperm proliferation (hypoplasia).

Scott et al. (1998) explain these observations in terms of the genomicimprinting (inactivation) of genes that contribute to endosperm vigour,either positively or negatively. Accordingly, paternal gametes have anoverall positive effect on endosperm growth because genes that inhibitendosperm growth or functionality are imprinted, whilst genes that havea positive effect escape imprinting and are active in the endosperm.Adding more paternal genomes into the endosperm via a tetraploid pollenparent therefore increases the number of stimulatory genes resulting ina larger endosperm. Maternal genomes have the opposite effect.Importantly, imprinting effects have been recorded in a wide range ofplant species including maize and brassicas. In mammals, a number ofgenes that influence fetal growth (typically expressed in the placenta)also exhibit uniparental expression due to imprinting duringgametogenesis. Extra doses of these genes also have dramatic effects onembryo size.

Hybridization is recognized as an important process for producingoffspring having a combination of desirable traits from both parents.Hybridization may be interspecific or intraspecific. Interspecifichybridization is used for introducing desirable traits such as diseaseresistance into crop species. However, the ability to make successfulsexual crosses is frequently restricted to closely related speciesbecause of the existence of a variety of pre-fertilization andpost-fertilization reproductive barriers (see Stoskopf, Tomes andChristie, 1993). One type of post-fertilization barrier is associatedwith poor or disrupted endosperm development (post-fertilizationendosperm development barrier), which results in non-viable seed (seeEhlenfeldt and Ortiz, 1995). Endosperm failure in unsuccessful crossesis due to the operation of a genetically determined system known asendosperm dosage (Haig and Westoby, 1991). Endosperm dosage is a form ofgenomic imprinting. The removal of the endosperm dosage barrier tosexual interspecific hybridization would have economic benefits, sincenon-sexual techniques for hybridization e.g. somatic hybridization arecostly and difficult.

The endosperm dosage system may also prevent intraspecific hybridizationwhere the parents are of different genomic constitutions (ploidies)(Haig and Westoby, 1991).

The occurrence of successful intra- and interspecific hybridization canalso be problematic. In particular, hybridization between geneticallymodified crop plants and non modified cultivated or wild plants therebycreating hybrids carrying transgenes with the potential forenvironmental and other damage inherent in this form of “transgeneescape”, has caused alarm within the public and the regulatoryauthorities.

There are various strategies that might be used to prevent transgeneescape from crops into the wider environment. Critically, a range orspectrum of methods should be available to meet practical constraintsimposed by the requirements of plant breeders and seed producers and thelife histories of specific crop species when in the hands of farmers.For example, the complete elimination of flowering is acceptable invegetable crops and forage grasses during the ‘cropping stage’, butunless this trait is conditional in some way, the production of seed bythe seed producer, or the breeding of new varieties by the plantbreeder, is rendered difficult or impossible.

In crops where the harvest is a fruit or a seed, given that most cropspecies are self-pollinating, the production of pollen, by at least themajority of flowers, is essential. Most of the major crops fall intothis category.

Cleistogamous plants produce flowers that develop normally but whichfail to open. Consequently, self pollination occurs, but no pollenescapes from the flower. Whilst this the implementation of this solutionwould ‘only’ require modifications to flower design, such as approachmight be criticized on the grounds that pollen could escape from damageflowers.

The production of viable sexual hybrids occurs within species(intra-specific hybridization) or between species (inter-specifichybridization). However, in the case of inter-specific hybridization, asuccessful outcome—viable hybrid seed—is usually only possible betweenclosely related species. Two main barriers prevent hybridization betweenmore widely diverged species—inter-specific incompatibility at thestigma surface or within the style, which prevent fertilization, andpost-fertilization barriers which cause seed abortion, usually throughfailures in endosperm development (Brink and Cooper, 1947; Ehlenfeldtand Ortiz, 1995).

Brink and Cooper (1947) working in Lycopersicum were the first topropose that the primary reason for failure of inter-specific crosseswas the same as for intraspecific crosses, namely failure of theendosperm itself. The operation of this type of barrier has beenreported in numerous species including the Brassicas (see Haig andWestoby, 1991). These authors and others (see Ehlenfeldt and Ortiz,1995) also proposed that endosperm failure in inter-specific crosses isdue to an effective, rather than actual, imbalance in the normal ratioof maternal to paternal genomes in the endosperm. Different species areproposed to have different genomic strengths. Hence a cross betweenplants of the same ploidy may fail because the relative genomicstrengths of their respective genomes result in a lethal effectivegenomic imbalance within the hybrid endosperm. Likewise a cross betweentwo plants of different ploidies may succeed provided their relativegenomic strengths result in a hybrid endosperm with a balanced genomicconstitution. The setting of genomic strength is proposed to involvegenomic imprinting, although the exact nature of the relationship is notunderstood.

In summary, the failure of intraspecific (interploidy) crosses andcrosses between species may have a common cause—a genomic imbalancewithin the endosperm mediated by genomic imprinting. Modifying thegenomic strength of one or both of a pair of species that normallyhybridize may have application in generating a lethal relative endospermimbalance, thereby creating a post fertilization barrier between the twospecies. The same approach may have application in providing apost-fertilization barrier within a species, for example betweengenetically-engineered crop varieties and non-engineered varieties.Practically, for transgene containment the genomic strength of the cropcould be modified to prevent cross hybridization with any problematicclose relatives. Such a technology would facilitate the exploitation ofgenetically modified plants, with considerable economic andenvironmental benefits.

There is currently considerable research effort to develop transgenictechnologies (see Koltunow et al., 1995) to introduce apomixis into cropspecies. In natural apomictic plant species 2n seed is produced withoutfertilization of the egg. The genetic constitution of the embryo istherefore identical to that of the seed parent. The economic benefits ofintroducing an apomixis system into crop species include true breedingF1 hybrids. Currently, F1 hybrid seed is produced annually byhybridizing two genetically distinct parents in a labor intensive andcostly process. True breeding (apomictic) F1 hybrids could be propagatedfor sale without the hybridization step. The removal of this step wouldpotentially therefore reduce production costs.

An essential aspect of apomixis is that the embryo is derived from acell with an unreduced (2n) number of chromosomes. In natural apomictsthis is achieved by modifying meiosis (meiotic reconstitution) such that2n gametes are produced, or deriving the embryo from a somatic cell withthe 2n number of chromosomes. Irrespective of the origin of the embryothe endosperm is invariably derived via meiosis which is eitherrestitutional or reductional. In the former case the two polar nuclei,which upon fertilization produce the endosperm, are 2n and in the latercase n. Given that natural apomicts utilize endosperms generated in thisway it is likely to be the case for genetically engineered apomicticcrop plants.

A potential problem in the development of apomictic crop species, giventhis likely dependency on ‘sexual endosperms’ (formed by fertilization),is ensuring the successful development of the endosperm, since theendosperm is required to nourish the embryo or itself represents theprincipal economic harvest. One barrier to endosperm development is theendosperm dosage system. In species with an endosperm dosage system theration of maternal to paternal genomes in the endosperm is 2:1.Deviation from this ratio results in endosperm abortion and seedlethality (Haig and Westoby, 1991). Natural apomicts have adopted anumber of strategies to ensure endosperm development. A few species(autonomous apomicts) develop a gynogenetic endosperm (maternal) in theabsence of fertilization of the polar nuclei. The majority however,retain fertilization of the polar nuclei and maintain a 2:1 genomicratio by modification of either male meiosis (to produce unreducedgametes) or the fertilization process e.g. fertilization involves only 1polar nucleus. Still other species successfully deviate from the genomic2:1 ratio.

For engineered apomixis the most attractive solution for ensuring theendosperm development is the provision of autonomous endospermdevelopment. Solutions involving fertilization of the polar nuclei arelikely to complicate the delivery of apomixis, for example bynecessitating the introduction of a mechanism to prevent fertilizationof the “egg” or the need to devise ways to produce 2n male gametes, orby some other means ensure a 2:1 genomic ratio.

One approach to developing an autonomous apomict involves the inductionand isolation of mutant genes that condition endosperm development insexual species without fertilization. Extensive screening efforts inArabidopsis met with limited success having identified several mutantgenes that condition only limited endosperm development in the absenceof fertilization (Ohad et al., 1996; Chaudhury et al., 1997; Ohad etal., 1999; Kiyosue et al., 1999; Luo et al., 1999). One potentialexplanation is that these mutations trigger endosperm development but donot overcome the effects of the endosperm dosage system. Endosperms inthe mutants would have a genetic constitution of 2 maternal:0 paternalgenomes, which deviates from the normal 2:1 genomic ratio.Significantly, Scott et al., 1998, recently showed that Arabidopsispossesses a dosage system capable of causing seed abortion where theratio of parental genomes in the endosperm deviates significantly from2:1.

Autonomous apomixes would enable the crop to produce seed without anyrequirement for pollen. Hence transgene escape through pollen could beprevented by arranging for the crop plant to carry any form of malesterility that stops the production or release of functional pollen.

The interploidy cross effect on seed size, the post-fertilizationendosperm development barrier to interspecific hybridization and thebarrier to autonomous endosperm development are all explicable in termsof genomic imprinting.

The interploidy cross effect on seed size, the post-fertilizationendosperm development barrier to interspecific hybridization and thebarrier to autonomous endosperm development are all explicable in termsof genomic imprinting.

In mammals, a number of genes that influence fetal growth (typicallyexpressed in the placenta) exhibit uniparental expression due to genomicimprinting during gametogenesis. Extra doses of these genes can havedramatic effects on embryo size (Solter, 1998). Genomic imprinting alsoprevents the development of gynogenetic or androgenetic (two parentalgenomes, no maternal genome) embryos (Solter, 1998).

In mammals, genes selected for imprinting are maintained in inactivestate by DNA methylation. The enzyme responsible is DNAmethyltransferase (MET) which is encoded by a single gene. Mice embryoscontaining an inactive DNA methyltransferase gene die at an earlydevelopmental stage and express both parental copies of genes that arenormally imprinted (i.e. uniparentally expressed) (Li et al., 1993).This demonstrates the involvement of DNA methyltransferase in genomicimprinting and a requirement for imprinting in normal development.

In plants the imprinting mechanism is unknown. However, plant genomescontain relatively large amounts of the modified nucleotide5-methylcytosine (Gruenbaum et al., 1981). Despite evidence implicatingcytosine methylation in plant epigenetic phenomena, such as cosupressionand inactivation of transposable elements (Napoli et al., 1990; Benderet al., 1995; Brutnell and Dellaporta, 1994, Martienssen et al., 1995;Matzke and Matzke, 1995) the role of cytosine methylation in plantdevelopmental processes and genomic imprinting remains unclear.

To date three different genes have been found that may be imprinted inthe maize endosperm: tubulin (Lund et al. 1995), a storage proteinregulator gene dzr (Chaudhuri, and Messing, 1994) and the r genetranscription factor that regulates anthocyanin biosynthesis (Kermicleand Alleman, 1990). In each case, the maternally inherited allele isundermethylated, over-expressed or both, whereas the paternallyinherited allele is more methylated or has a reduced level ofexpression.

In Arabidopsis, ddm mutants (decrease in DNA methylation) have beenisolated with reduced levels of cytosine methylation in repetitivesequences, although the mutations do not result in any detectable changein DNA methyltransferase activity (Vongs et al, 1993; Kakutani, 1995).After several generations of self pollination, ddm mutants exhibit aslight delay (1.7 days) in flowering, altered leaf shape, and anincrease in cauline leaf number (Kakutani et al., 1995). Repeated selfpollination of ddm mutant plants does however result in the appearanceof severe developmental abnormalities (Kakutani et al., 1996).

Arabidopsis plants expressing DNA methyltransferase 1 (Met1) antisense(Met las) gene contain reduced levels of DNA methyltransferase activityand a correspondingly reduced level of general DNA methylation (Finneganet al., 1996; Ronemus et al., 1996). In contrast to ddm mutants,Arabidopsis plants expressing a Met1as gene develop variousdevelopmental abnormalities at high frequency and without repeatedself-fertilization, including floral abnormalities (Finnegan et al.,1996). PCT/US971/13358 also reports that Arabidopsis plants expressing aMet1as gene alter the rate of development of the plant. The developmentof the endosperm in ddm mutants and plants expressing Met las has notbeen reported.

The present invention is based on the unexpected observation that adecrease of about 90% in the amount of methylated DNA present in a plantgenome results in the production of gametes, both male and female, thatbehave in a manner that is consistent with the removal or attenuation ofgenomic imprinting. This is exemplified by the following experiments:

1. Endosperm development in seeds derived from a cross between a wildtype 2x plant, as seed parent, and a 2x Met1as plant as pollen parent(2x-2xMet1as), resembles endosperm development in seeds derived from a4x-2x interploidy cross (FIGS. 1 and 3).—the endosperm issmall/underdeveloped. The resulting seed is smaller in weight terms thanseed from control 2x-2x crosses (Table 1). Hence the male gametes from aMet1as plant behave like a female gamete from a wild type plant. Thiscan be explained by proposing the removal or attenuation of imprintingin the male gamete.

2. Endosperm development in seeds derived from a cross between a2xMet1as plant, as seed parent, and a wild type 2x plant as pollenparent, strongly resembles endosperm development in seeds derived from a2x-4x interploidy cross between wild type plants (FIGS. 1 and 3).—thatis, the endosperm is large/overdeveloped. The resulting seed is largerin weight terms than seed from control 2x-2x crosses (Table 1). Hencethe female gametes from a 2xMet1as plant behave as a male genome of anormally methylated diploid plant. This can be explained by proposingthe removal or attenuation of imprinting in the female gamete.

3. Reciprocal crosses between 2xMet1as and 4x wild type plants result inseed abortion (FIGS. 1 and 3); consequently seeds derived from thesecrosses are shriveled and do not germinate (Table 1). The behavior ofthe endosperm in seed generated in these crosses depends on thedirection of the cross. Where the 4x plant is the seed parent theendosperm is extremely under-developed and contains very few endospermnuclei and a very small chalazal endosperm (FIG. 1, Table 1). Incontrast, where the 4x plant is the pollen parent the endosperm of theresulting seeds is over-developed, and contains many endosperm nucleiand a very well developed chalazal endosperm with many associatedchalazel nodules (FIGS. 1 and 3, Table 1). This outcome resembles thoseobtained in crosses between 2x and 6x wild type plants which routinelyfail to produce viable seed (FIG. 3) and display very under- (6x-2x) orover-developed (2x-6x) endosperm depending on the direction of thecross. These crosses represent examples of lethal parental genomicexcesses within the endosperm that result from the large disparitybetween the ploidy level of the respective parents. The similaritybetween the outcomes and the behavior of the endosperm in 2xMet1as—4xand 2x-6x reciprocal crosses can be explained by proposing that male andfemale gametes derived from 2xMet1as plants behave, in part, likegametes of the opposite sex with respect to genomic imprinting. Thisagain strongly suggests that DNA hypomethylation caused by the MET1asgene removes or strongly attenuates genomic imprinting.

4. The behavior of plants homozygous for the ddm mutation in reciprocalcrosses with 2x and 4x wild type plants is very similar to that ofplants homozygous for the Met1as gene (see FIG. 2 and Table 1). Thisstrongly suggests that the basis of the interploidy cross effect isassociated with general DNA hypomethylation.

BRIEF SUMMARY OF THE INVENTION

Thus, in a first aspect, the present invention provides a method for theproduction of modified endosperm which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression in the male or female germ line and/or gametes ofthe resultant plant and one or more sequences whose expression ortranscription product(s) is/are capable of modulating genomicimprinting.

As will be described herein, modulation of imprinting of plant gameteDNA can be used after endosperm development. The effects can be appliedto male or female gametes of the transformed plant. Thus, in a secondaspect, the present invention provides a method for the production ofmodified endosperm which comprises the step of transforming a plant, orplant propagating material, with a nucleic acid molecule comprising oneor more regulatory sequences capable of directing expression within thedeveloping gynoecium, especially the cell lineage that gives rise to thefemale germ line (megasporocyte tissue), within the ovule of theresultant plant and one or more sequences whose expression ortranscription product(s) is/are capable of modulating genomicimprinting.

In a third aspect, the present invention provides a method for theproduction of modified endosperm which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression within the developing stamen, especially the celllineage that gives rise to the male germ line (microsporocyte tissue) ofthe resultant plant and one or more sequences whose expression ortranscription product(s) is/are capable of modulating genomicimprinting.

There are a number of proteins known or suspected to be involved in theprocess of genomic imprinting. Altering the rate of expression of thosegenes in the germ line of either sex can also be used to alter thedevelopment of the endosperm in a parent-specific manner.

In the African claw toad Xenopus laevis, the product of themethyl-cytosine binding protein 2 (MeCP2) has been showing tospecifically bind to methylated cytosines (Kass et al., 1997; Jones etal., 1998). This protein, of which conserved homologs in mammals alsoexist, forms a complex at the C-met locus with several other proteins.Amongst these are the transcription-repression mSin3 proteins (Nan etal., 1998; Laherty et al., 1997) and a number of histone process ofanchoring histones to the DNA and hence the formation of heterochromatinand the silencing of genes (reviewed in Razin, 1998 and Pazin andKadonaga, 1997). The MeCP2-protein may thus constitute the first step inthe gene silencing process by guiding the heterochromatin-formingmachinery to C-met loci. Interestingly, in contrast with this theprotein has also been found to have a de-methylating function in that itremoves methyl-groups from cytosine residues (Bhattacharya et al.,1999).

If the homologs of proteins in the C-met binding complex in plants arelikewise involved in uniparental gene silencing (imprinting) theninactivation of these genes in the maternal or paternal germ lines wouldbe predicted to mimic the uniparental inactivation of the genesresponsible for methylation. In addition, there could be a cumulativeeffect if more than one gene is inactivated. If for instanceinactivation of the MET1 gene by antisense transcription or ds-RNA inone of either germ lines is not complete, then introduction of anadditional vector causing inactivation of one of the other components ofthe imprinting machinery will enhance the effect.

In a preferred aspect, the present invention provides a method for theproduction of modified endosperm based on targeting the germ line orgametes with transgenes which alter the capacity of genes to form,maintain or express imprints. This can be achieved in a number of ways.Firstly, by incorporation of one or more sequences encoding proteinsassociated with the application or maintenance of genetic imprints.Specifically, such sequences may encode a histone deacetylase, methylcytosine binding protein or Sin 3 proteins, for example, m Sin 3.

Alternatively, the transgene may incorporate sequences including the FIEgene or the FIS gene, for example fis1, fis2 or fis3.

Imprinted genes may also contain, or be located close to, signals withinthe DNA sequence (a particular nucleotide sequence motif) that mark themout for imprinting during gamete production. Such a motif may, inaddition to expressed proteins associated with the formation and/ormaintenance of genomic imprints, be involved in the formation of an“imprinting complex”. It is contemplated that removing or inactivatingthe DNA motif, or restricting the availability of the associatedproteins, in the imprinting complex may provide a means for preventingor attenuating the application of imprints, thereby allowing theexpression of genes which may otherwise be silenced in the endosperm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention further provides methods for removing orattenuating genomic imprinting, based on targeting the germ line orgametes with transgenes-which alter the methylation pattern of genes, ortheir capacity to form or maintain imprints, within the developingendosperm. Thus, in a fourth aspect, the present invention provides amethod for the production of modified endosperm, which comprises thestep of transforming a plant, or plant propagating material, with anucleic acid molecule comprising one or more regulatory sequencescapable of directing expression in the male or female germ line and/orgametes of the resultant plant, and one or more sequences whoseexpression or transcription product(s) is/are capable of altering thedegree of methylation of nucleic acid.

The restriction of imprint removal or attenuation to one or other sex ofgamete is desirable for 3 reasons:

1. To provide for removal of imprinting in a single sex of gamete withinan individual plant. This will produce the asymmetry of imprinting thatis required to mimic the interploidy cross effect in a self-fertilizingplant.

2. To prevent developmental abnormalities that are associated withgeneralized hypomethylation, such as occurs with the CaMV35S driven Met1antisense gene.

3. To prevent the attenuation of the interploidy cross effect due to theexpression of the hypomethylation gene (Met1as) within the endosperm.Crosses between two 2xMet1as plants result in seed with a slightlyincreased number of endosperm nuclei and normal seed weight (Table 1),which is most easily explained by proposing that the combination ofhypomethylated gametes of both sexes allows normal endospermdevelopment.

The important property of the nucleic acid molecules used in thetransformation step is that DNA of cells that contribute to one sex ofgerm line is subject to alteration of the pattern of DNA methylationthrough the activity of the transgenes. The germ-line is the tissuewithin the reproductive organs that produces the gametes. In the anthers(stamen) this is the microsporogenous cell tissue and in the pistil(gynoecium) the megasporocyte tissue.

Since the timing of the application of the genomic imprints is currentlynot known the activity of the regulatory sequences, e.g. promoters (orfragments of promoters) promoters should be as broad as possible whilstremaining consistent with the principles discussed herein.

As will be described herein, alteration of the methylation of plantgamete DNA can be used to modify endosperm development. Thus, in a fifthaspect, the present invention provides a method for the production ofmodified endosperm, which comprises the step of transforming a plant, orplant propagating material, with a nucleic acid molecule comprising oneor more regulatory sequences capable of directing expression within thedeveloping gynoecium, especially the cell lineage that gives rise to orcomprises the female germ line (megasporocyte tissue), within the ovuleof the resultant plant, and one or more sequences encoding one or moreproteins which cause methylation or demethylation of nucleic acid.

In this aspect of the invention, the resultant endosperm is larger, andthe seed produced is heavier. Herein, suitable promoters includepromoter fragments from the Arabidopsis AGL5 gene (Sessions et al.,1998), the Petunia FBP7 and FBP11 genes (Angenent et al., 1995; Colomboet al., 1995), Arabidopsis BEL1 gene (Reiser et al., 1995) ArabidopsisMEDEA (FIS1) gene (Grossniklaus et al, 1998; Kiyosue et al., 1999),Arabidopsis FIS2 (Kiyosue et al., 1999), FIE (FIS3) (Ohad et al., 1999;Kiyosue et al., 1999), orthologs/homologues of these genes from otherspecies; Other promoters that drive expression that is restricted tocells within the female reproductive organs that contribute to thefemale germ line would also be suitable. Especially suitable arepromoters from gynoecium-specific genes that are first expressed duringearly gynoecium development, preferably before the differentiation ofindividual ovules, and which maintain their expression until ovuledifferentiation is complete (contain egg cell and binucleate centralcell).

As used herein, the term “homologues” of the genes is defined to includenucleic acid sequences comprising the identical sequence to the gene ora sequence which is 40% or more identical, preferably though 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% to the sequence of the geneat the nucleic acid residue level, using the default parameters of theGAP computer program, version 6.0 described by Deveraux et al., 1984 andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch 1970 as revised by Smith and Waterman 1981.

In a sixth aspect, the present invention provides a method for theproduction of modified endosperm which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression within the developing stamen, especially the celllineage that gives rise to or comprises the male germ line(microsporocyte tissue) of the resultant plant and one or more sequencesencoding one or more proteins which cause methylation of demethylationof nucleic acid.

In this aspect of the invention, the resultant endosperm is smaller, andhence the seed is lighter. Herein, suitable promoters include promoterfragments derived from the Arabidopsis genes APETALA3 (Jack et al.,1992; Irish and Yamamoto, 1995), the Arabidopsis PISTILATTA gene (Gotoand Meyerowitz, 1994), the Arabidopsis E2 (Foster et al., 1992), theArabidopsis APG (Roberts et al., 1993), homologues/orthologs of thesegenes from other species. Other promoters that drive expression that isrestricted to cells within the male reproductive organs that contributeto the male germ line would also be suitable. Especially suitable arepromoters from stamen-specific genes that are first expressed duringearly stamen development, preferably before the differentiation ofindividual microsporocytes, and which maintain their expression untilstamen differentiation is complete.

Herein, promoters that drive gene expression in cells of the germ lineor in cells that represent the direct progenitors of the germ linewithin either the stamen or pistil and which, when in conjunction withthe Met1as gene, produce hypomethylated gametes are referred to as ‘germline’ promoters.

Thus, as will be appreciated by the skilled person, the presentinvention allows for the modification of the endosperm such that it iseither increased or decreased in size. In addition, the development ofthe endosperm can be altered such that the modified plants can be usedin carrying out intraspecific hybridization, erecting artificialbarriers to intra- and interspecific hybridization to prevent “transgeneescape”, or in engineering apomixes.

In one specific embodiment, the degree of methylation is increased. Thiscan readily be achieved by incorporating one or more sequences encodingone or more methylating enzymes into the transgene.

Examples of suitable methylating enzymes include:

i) Methylase 1 (acc. nr. L10692);

ii) Methylase I-like gene (acc. nr. Z97335);

iii) Methylase 2 (acc. Nr AL021711); and

iv) Chromomethylase (acc. Nr. U53501);

all from Arabidopsis.

In another specific embodiment, the degree of methylation is decreased.This can be achieved in a number of ways. Firstly, by incorporation ofone or more sequences encoding one or more demethylating enzymes, suchas de-methylase (=MeCP2-homologue; see below) (acc. nr. AL021635) intothe transgene. Alternatively, the transgene can incorporate sequenceswhich cause down regulation of methylating enzymes already present inthe plant. For instance, one can use antisense sequences, e.g. theMet1as “gene”. In addition, it has been found that incorporation ofwhole or partial copies of an already present gene can result insuppression of gene expression. Thus, the transgene can incorporateadditional copies, or partial copies, of genes encoding methylatingenzymes already present in the plant. In another alternative, thetransgene can incorporate a sequence encoding a ribozyme.

With respect to the sequence, or sequences capable of altering thedegree of methylation, sequences encoding methylating or demethylatingenzymes can be used. Examples of the latter include:

i) Methylase 1-like gene (acc. nr. Z97335);

ii) Methylase 2 (acc. nr. AL021711);

iii) Chromomethylase (acc. nr. U53501);

iv) de-methylase (=MeCP2-homologue; see below)(acc. nr. AL021635);

In Arabidopsis, possible homologs of the following genes have beenfound:

MeCP2 (acc. nr. AL021635)

HDAC1/2 (acc. nr. AF014824 & AL035538)

mSIN3 (acc. nr. AC007067_(—)5 & AC002396)

_p300: a histone acetylation-gene (acc. nr. AC002986.1 & AC002130.1)

In a seventh aspect, the present invention provides an isolated orrecombinant nucleic acid molecule, eg a DNA molecule, which comprisesone or more regulatory sequences capable of directing expression in themale or female germ line and/or gametes of a plant and one ore moresequences capable of altering the degree of methylation of nucleic acid.

In a preferred embodiment of the seventh aspect, the degree of nucleicacid methylation is decreased. An eight aspect of the present inventionprovides the use of a transgene in which the degree of nucleic acidmethylation is decreased, as a post-fertilization barrier tohybridization, for example, interspecific or intraspecific hybridizationbetween plants.

The expression “barrier” is defined to include all forms of reproductivebarrier which are associated with poor or disrupted endospermdevelopment. Specifically, the term barrier refers to apost-fertilization endosperm development barrier, which results innon-viable seed.

The transgene provides a barrier to hybridization by modifying thegenomic strength of one or both a pair that normally hybridize therebycausing an effective genomic imbalance leading to failed or disruptedendosperm development. The genomic strength may be modified by removingor attenuating genomic imprinting through DNA hypomethylation. Theadvantage of preventing hybridization between plants of the same species(interspecific hybridization) is discussed earlier in the application inthe context of preventing transgene escape.

In a ninth aspect, the present invention provides the use of a transgenein which the degree of nucleic acid methylation is decreased, inovercoming a post-fertilization barrier to hybridization. In thiscontext, the barrier to hybridization between plants of the same species(interspecific hybridization) arises through endosperm dosage whichleads to failed endosperm development. The removal or attenuation ofgenomic imprinting through DNA hypomethylation, may remove the endospermdosage barrier to interspecific hybridization. The removal of theendosperm dosage barrier to several interspecific hybridization wouldhave economic benefits as discussed previously in the application.

The nucleic acid of the seventh aspect of the invention will normally beemployed in the form of a vector and such vectors form a further aspectof the invention.

The vector may be for example a plasmid, cosmid or phage. Vectors willfrequently include one or more selectable markers to enable selection ofcells transfected or transformed and to enable the selection of cellsharboring vectors incorporating heterologous DNA. Examples of such amarker gene include antibiotic resistance (EP-A-0242246) andglucuronidase (GUS) expression (EP-A-0344029). Expression of the markergene is preferably controlled by a second promoter which allowsexpression in cells other than the gametes, thus allowing selection ofcells or tissue containing the marker at any stage of regeneration ofthe plant. The preferred second promoter is derived from the gene whichencodes the 35S subunit of Cauliflower Mosiac Virus (CaMV) coat protein.However any other suitable second promoter could be used.

Cloning vectors may be introduced into E. coli or another suitable hostwhich facilitates their manipulation. DNA in accordance with theinvention will be introduced into plant cells by any suitable means.Thus, according to yet a further aspect of the invention, there isprovided a plant cell including DNA in accordance with the invention.

DNA may be transformed into plant cells using a disarmed Ti-plasmidvector and carried by agrobacterium by procedures known in the art, forexample as described in EP-A0117618 and EP-A-0270822. Alternatively, theforeign DNA could be introduced directly into plant cells using aparticle gun. This method may be preferred for example when therecipient plant is a monocot.

A whole plant can be regenerated from a single transformed plant cell,thus in a further aspect the present invention provides transgenicplants (or parts of them such as propagating material) including DNA inaccordance with the invention. The regeneration can proceed by knownmethods. When the transformed plant flowers it can be seen to be malesterile by the inability to produce viable pollen. Where pollen isproduced it can be confirmed to be non-viable by the inability to effectseed set on a recipient plant.

The present invention also provides transgenic plants and the sexualand/or asexual progeny thereof which have been transformed with arecombinant DNA sequence according to the invention. The regeneration ofthe plant can proceed by any known convenient method from suitablepropagating material.

A further aspect of the present invention provides a method formanipulating genomic imprinting in a plant, which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression in the male or female germ line and/or gametes ofthe resultant plant, and one or more sequences whose expression ortranscription product(s) is/are capable of altering the degree ofmethylation of nucleic acid.

Preferred features for each aspect of the invention are as for eachother aspect mutatis mutandis.

The present invention will now be described with reference to thefollowing examples, which should not be construed as in any way limitingthe invention. The examples are accompanied by the following figures.

BRIEF SUMMARY OF THE DRAWINGS

FIG. 1—Embryo and endosperm development following crosses withmet1-antisense expressing plants as a parent. Confocal micrographs ofFeulgen-stained seeds 4-6 days after pollination. Column 1, embryo;column 2, chalazal endosperm; column 3, peripheral endosperm. Note apaternal excess phenotype (over developed chalazal endosperm, highlyproliferated peripheral endosperm) in crosses with a demethylated plantas the mother (row 1, 2) and a maternal excess phenotype (small orabsent chalazal endosperm and a poorly developed peripheral endosperm)in crosses with a demethylated plant as the father (row 4, 5). See textfor full details.

FIG. 2—Embryo and endosperm development following crosses withddm1-mutant plants as a parent. Confocal micrographs of Feulgen-stainedseeds 4-6 days after pollination. Column 1, embryo; column 2, chalazalendosperm; column 3, peripheral endosperm. See text for full details.

FIG. 3—Embryo and endosperm development following interploidy crossesand balanced crosses. Confocal micrographs of Feulgen-stained seeds 4-6days after pollination. Column 1, embryo+peripheral endosperm; column 2,chalazal endosperm. For the 2x-4x and 2x-6x crosses (row 6, 7) theperipheral endosperm is shown as an inset. See text for full details.

FIG. 4—Schematic diagram showing the method of construction ofpAGL5-bin.

FIG. 5—Schematic diagram showing the method of construction of pAP3-bin.

FIG. 6—Schematic diagram showing the method of construction of pAGL5-asMET1.

FIG. 7—Schematic diagram showing the method of construction ofpAP3-asMET1.

FIG. 8—Seed production following inter-specific crosses betweenArabidopsis thaliana and Arabidopsis lyrata. Light micrographs of seedstaken from mature seed pods. A, 4x, A. thalianaX a. lyrata; note seedsare shriveled (see Table 3 for germination data). B, 4x A. thaliana Met1a/s×A. lyrata (4x A. thaliana Met1a/s=hypomethylated tetraploid lineexpressing Met1 a/s gene; note that seeds are plump (see Table 3 forgermination data). See text for full details.

FIG. 9—Seed production following inter-specific crosses betweenArabidopsis thaliana and Cardaminopsiss arenosa. Light micrographs ofseeds taken from mature pods. A, 4x A. thalianaXC. arenosa; note seedsare plump (see Table 3 for germination data).

FIG. 10—Seeds from afie-1/FIE×FIE/FIE cross. (A) Light micrographshowing the two classes of seeds, plump (pl) and shriveled (sh).(Bar=5mm). (B-G) Confocal micrographs of normal (B-D) and aborting (E-G)seeds at 8 DAP, centered on micropylar (B, E), central (C, F), andchalazal (D, G) regions of the embryo sac. The endosperm in (E-G) isovergrown and has not cellularized. Bar=50 μm.

FIG. 10—Seeds from a fie-1/FIE×FIE/FIE cross. (A) Light micrographshowing the two classes of seeds, plump (pl) and shriveled (sh). (Bar=5micrometers). (B-G) Confocal micrographs of normal (B-D) and aborting(E-G) seeds at 8 DAP, centered on micropylar (B, E), central (C, F), andchalazal (D, G) regions of the embryo sac. The endosperm in (E-G isovergrown and has not cellularized. Bar=50 micrometers.

FIG. 11—Seeds from a [fie-1/FIE×FIE/FIE; MET1 als/MET1 a/s] cross. (A)Light micrograph showing the two classes of seeds. All seeds are plump,indicating that a pollen parent hypomethylated by the MET1 a/s transgenecan rescue fie-1 mutant seeds. Bar=5 mm. (B) Identification of the fie-1and FIE alleles by PCRA and restriction enzyme analysis. The wild typeFIE allele produces four bands (lane 1, WT) while fie-1/FIE heterzygotes(lane 3, Het) have an extra band. All large seeds scored had theheterozygous pattern (lane 3) while all small seeds were wild type (lane4). (C-H) Confocal micrographs of seeds at 8 DAP. The seed in (C-E) hasa similar phenotype to seeds from interploidy crosses generatingmaternal genomic excess, while (F-H) shows characteristics of paternalexcess (see text, and Scott et al., 1998). Bar=50 μm.

FIG. 12—Autonomous endosperm development in unfertilized seeds ofArabidopsis thaliana.

Confocal micrographs of fertilization-independent seeds produced byemasculated fie-1/FIE heterozygotes with normal and reduced methylation.(A-C) Seed-like structure from a plant with normal methylation. (A)Optical section showing peripheral endosperm but no well differentiatedchalazal endosperm. Bar=50 μm. (B) Clustered endosperm nuclei atperiphery. (PE, peripheral endosperm.) Bar=50 μm. (C) Endosperm atmicropylar (MP) and chalazal (CHP) poles. (D-G) Seed-like structuresfrom fie-1/FIE; MET1 a/s plants. (D, E) Type 1 seed-like structures at 7(D) and 10 (E) days after emasculation (DAE). In these the endospermcellularizes and fills the interior of the embryo sac. (F, G) Type 2seed-like structures at 7 (F) and 10 (G) DAE. These produce micropylarand chalazal in addition to peripheral endosperm.

EXAMPLES Example 1

The use of gametes from hypomethylated plants (Met1as and ddm) mimicsthe interploidy cross effect (alters number of endosperm nuclei formedand consequently the weight of mature seed).

Reciprocal interploidy (different ploidy) crosses between diploid (2x),and tetraploid (4x) (Scott et al., 1998) or hexaploid (6x) (Scott etal., 1998) Arabidopsis plants result in changes to both the size of theendosperm, in terms of the number of endosperm nuclei and volume of thechalazal endosperm, and to the dry weight of mature seeds (see Table 1)and the viability of the seed (Table 1). This is the interploidy crosseffect.

Crosses Involving Met1as Plants

Intraploidy (same ploidy) crosses between 2x Met1as plants and 2x wildtype plants mimic this effect (see Table 1 and FIGS. 1 and 3). A crossbetween a 2x Met1as plant as seed parent and a 2x wild type plant aspollen parent produces seeds with an average of 450 endosperm nuclei (anincrease of 130% over 2xmet-2xmet cross), a relative increase inchalazal endosperm volume of 75% compared to 2xmet-2xmet seed, and amature dry weight of 20 μg (an increase of 33% compared to seed from2xmet-2xmet cross) (see Table 1).

A cross between a 2x wild type plant as seed parent and a 2x Met1asplant as pollen parent produces seeds with an average of 200 endospermnuclei (a reduction of 43% over 2xmet-2xmet cross), a relative decreasein chalazal endosperm volume of 50% compared to 2xmet-2xmet seed, and amature dry weight of 10 μg (a decrease of 30% over a wild type2xmet-2xmet cross) (see Table 1). TABLE 1 Outcomes of control crossesand crosses involving Met1 antisense and ddm mutant plants. Maximumnumber of Relative Relative Interploidy Viability of peripheral volumeof change to Seed cross hybrid see endosperm chalazal cellularizationweight Cross phenotype¹ (%)² nuclei³ endosperm⁴ time (days)⁵ (μg)⁶ 2x-2xNA 95-100 400 1 0 22 4x-4x NA 95-100 400 2.5 0 36 6x-6x NA 95-100 3003.5 0 44 2x-4x PE 95-100 640 2 +1 54 4x-2x ME 95-100  80 0.6 −1 14 2x-6xPE 0⁷ 400 6.8 Absent 6 6x-2x ME 0⁷  50 0.2 −1.5 4 2xmet- PE 95-100 350 10 15 2xmet 2xmet- (90)⁸  (598)⁸ (13.6)⁸ 2xmet 2x-2xmet ME 95-100 200 0.5−0.5 10 2x-2xmet (93)⁸  (227)⁸ (9.5)⁸ 2xmet-2x PE 95-100 450 1.75 +0.520 2xmet-2x (97)⁸  (1,365)⁸   (32.5)⁸ 2xddm- PE 95-100 350 1.25 0 192xddm 2x-2xddm ME 95-100 250 0.5 −0.5 12 2xddm-2x PE 95-100 400 2 +0.521 4x-2xmet ME 0⁷ 740 4.4 >+3 15 4x-2xddm ME 0⁷ 150 0.3 −1.5 5 2xddm-4xPE 0⁷ 680 3.5 >+3 5NA, not applicable;PE, paternal excess;ME, maternal excess.¹either paternal (PE) or maternal (ME) excess as defined in Scott etal., 1998.²determined by germination on soil.³counts done as described in Scott et al., 1998.⁴calculated relative to amount in 2x-2x control cross at heart stage(approx. 5 DAP).⁵expressed relative to 2x-2x control cross (usually 5 DAP).⁶measured as described in Scott et al., 1998.⁷seeds shriveled.⁸this experiment was performed subsequent to the experiment that yieldedthe non-bracketed data and used improved growing techniques for themet1a/s plants. This resulted in more vigorous plants which presumablyaccounts for the observed changes in seed weight. Note however that thechanges are qualitatively the same as the original experiment i.e.,2x-2xmet are smaller than 2xmet-2xmet and 2xmet-2x are larger.

The present and (possible) activity of the Met1a/s gene within theendosperm potentially complicates the interpretation of the dataproduced in out crosses involving homozygous Met1a/s plants. In suchcrosses the endosperm (and embryo) inherit a single copy of the Met1as,either from the seed or pollen parent. If the Met1as is active withinthe endosperm it may,

-   1. disrupt endosperm development since Met1as plants show various    vegetative and floral abnormalities associated with the    mis-expression of certain genes that regulate development (Finnegan,    1996). However, the presence of the Met1as gene does not appear to    have this effect since the endosperms of seeds derived from self    pollinated Met1as plants appear developmentally normal except for a    degree of paternal excess (FIG. 1).-   2. attenuate the magnitude of the interploidy cross effect, by    demethylating and thereby erasing imprints from the genome    contributed by the normally methylated parent. The imprints must be    maintained and propagated in the endosperm if the interploidy cross    effect is to be mimicked. The removal of imprints via the action of    the Met1as gene could reactivate imprinted loci such that the    endosperm genomes behave as if derived from same ploidy parents.

To demonstrate that the interploidy cross effects described above aredue to the effect of the Met1a/s gene on the imprinting of gametesrather than any effect within the endosperm we present data from crossesinvolving plants hemizygous (that is carrying a single copy) of theMet1as gene. Such plants show patterns of general DNA demethylationsimilar to homozygotes. Hence gametes derived from these plants aregenerated in a hypomethylating environment, but because the plants arehemizygous only 50% of these gametes contain the Met1as gene. Thisenables gametes to be produced in a demethylating environment which thendo not subsequently contribute as Met1as into the endosperm when used incrosses. This allows the effect of removing imprints within the gametesto be evaluated in endosperms that do not contain the Met1as gene.

The results of reciprocal crosses involving hemizygotes and 4x wild typeplants are shown in Table 2. Both crosses result in a 1:1 ratio ofplump, viable:shriveled, inviable seed. The shriveled seeds are assumedto result from lethal parents excess caused by the union of ahypomethylated gamete from the hemizygote and a 2x gamete from the 4xparent. Conversely, the plump seeds are assumed to result from normallymethylated gamete from the hemizygote and a 2x gamete from the 4xparent. Met1as plants appear therefore to produce both normallymethylated and hypomethylated gametes. The plump seeds produce plantswhich segregate 1:1 for the Met1as gene. Presumably, the shriveled seedsalso segregate 1:1 for the Met1as gene. This data therefore demonstratesthat the presence of the transgene in the endosperm is not responsiblefor the lethality phenotype associated with 2xMet1as-4x reciprocalcrosses. If this were the case, seeds containing the Met1as gene wouldnot be recovered among the plump, viable seed class.

Crosses Involving ddm Mutant Plants

Table 1 shows that crosses between wild type diploid and wild typetetraploid plants and plants homozygous for the ddm mutation have verysimilar outcomes to crosses involving plants containing the Met1as gene.The common feature of the ddm mutation and the action of the Met1as geneis that plants containing these genes have highly hypomethylated DNA.This shows that the interploidy cross effect produced in crossesinvolving gametes derived from ddm and Met1as plants is related to DNAhypomethylation.

The hemizygote data (Table 2) further suggests that the phenomenoninvolves hypomethylation of the gametes, presumably through the removalof genomic imprints. TABLE 2 Outcomes of reciprocal crosses betweenArabidopsis plants hemizygous for the Met1as gene and wild type 4xplants. Mature Seed Seed Proportion phenotypes viability viable seedsSeed weight (%)¹ (%)² carrying (μg)⁴ Plump Shrivelled Plump ShrivelledMet1as gene Plump Shrivelled seeds seeds seeds seeds (%)³ seeds seeds4x-2xmetHET 50 50 95-100 0 50 11 2 2xmetHET-4x 50 50 95-100 0 50 23 8Abbreviations:2x, wild type diploid plant;4x, wild type tetraploid plant;2xmetHET, plant hemizygous for the Met1as gene¹scored by eye.²determined by germination on soil of seed from mature pods.³determined by PCR analysis on plants germinated from plump seeds.⁴measured as described in Scott et al., 1998.

Example 2

Construction of expression cassettes that restrict gene expression toeither the gynoecium or the stamen.

Example 1 demonstrates that uniparental demethylation can be used tocontrol seed size. However, the increase in seed weight in the cross2xmet1a/s-2x is smaller than for the corresponding interploidy cross(2x-4x). This may be due to the reduced fitness of the 35SMet1as femalelines since demethylation is approximately constitutive. In order toreduce and eliminate this effect and to allow seed size changes to beobtained in a single plant it is necessary to restrict demethylation asmuch as possible to the germ line or gametes.

a. Designing a General Female-Germ Line Specific Expression Vector

An expression vector based on the female-specific AGL5 promoter(Sessions et al. (1998)) is constructed as described below. The nospolyA signal sequence is excised from pCaMVNEO (Fromm et al. (1986)) asa BamHI, Hind III fragment and cloned between the BamHI and HindIIIsites of pBin19 (Bevan 1994) forming pNosterm-bin. A 2.2 kb AGL5promoter is PCRed from Arabidopsis genomic DNA using the primers AGL5Fand AGL5R which introduce an EcoRI and a KpnI site at the ends of theAGL5 PCR fragment. (SEQ ID NO:1) 5′ CCGAATTCTTCAAGCAAAAGAATCTTTGTGGGAG3′ AGL5F      EcoRI (SEQ ID NO:2) 5′ CGGTACCTATAAGCCCTAGCTGAAGTATAAACAC3′ AGL5R      KpnI

The AGL5 PCR fragment is cloned as an EcoRI, KpnI fragment between theEcoRI and KpnI sites of pNosterm-bin forming pAGL5-bin (FIG. 4).

b. Designing a General Male-Germ Line Specific Expression Vector

An expression vector based on the male-specific AP3 promoter (Irish andYamamoto (1995)) is constructed as described below. A 1.7 kb AP3promoter is PCRed from Arabidopsis genomic DNA using the primers AP3Fand AP3R which introduce an EcoRI and a KpnI site at the ends of the AP3PCR fragment. (SEQ ID NO:3) 5′ CCGAATTCAAGCTTCTTAAGAATTATAGTAGCACTTG 3′AP3F      EcoRI (SEQ ID NO:4) 5′ GGGTACCTTCTCTCTTTGTTTAATCTTTTTGTTGAAGAG3′ AP32      KpnIThe AP3 PCR fragment is cloned as an EcoRI, KpnI fragment between theEcoRI and KpnI sites of pNosterm-bin forming pAP3-bin (FIG. 5).

Example 3

Construction of chimaeric gene fusions between the female (Example 2a)and male (Example 2b) germ-line specific cassettes and the Met1antisense gene.

Expression of the MET1 gene can be reduced in the female or male germlines by employing techniques known in the art. For example MET1down-regulation can be achieved by expressing antisense MET or antisenseMET1 fragments or sense MET1 or partial sense MET1 or ribozymes directedagainst MET1 or combination of the preceding, from promoters expressedin the required germ-line. Below is an example of an antisense MET1approach.

a) The Construction of a Female Germ-Line Specific Met1as Gene

The MET1 cDNA is 4.7 kb long and is isolated by RT-PCR from ArabidopsiscDNA using the primers MET1F and MET1R. (SEQ ID NO:5)5′ACTCGAGATTTTGAAAATGGTGGAAAATGGGGC 3′ MET1F      XhoI (SEQ ID NO:6)5′ACCCGGGTGGTTATCTAGGGTTGGTGTTGAGGAG 3′ MET1R      SmaI

The resulting MET1 PCR fragment is then cloned as a SmaI, XhoI fragmentbetween the SmaI and Sail sites of pAGL5-bin forming pAGL5-asMET1 (FIG.6).

b) The Construction of a Male Germ-Line Specific Met1as Gene

The MET1 PCR fragment is cloned as a SmaI, XhoI fragment between theSmaI and SalI sites of pAP3-bin forming pAP3-asMET1 (FIG. 7).

Example 4

Introduction of female and male germ-line specific demethylating genesinto transgenic plants.

Chimaeric genes were introduced via Agrobacterium-mediatedtransformation into wild type diploid Arabidopsis using well knowntechniques.

a) pAGL5Met1as

Transgenic Arabidopsis plants containing the pAGL5Met1as gene werevegetatively normal and produced flowers with the normal complement offloral organs. Arabidopsis containing pAGL5Met1as were pollinated withpollen from wild-type diploid plants or allowed to self pollinate.Endosperm development in the resulting seeds was monitored by confocalmicroscopy (Scott et al., 1998) and seed weights were measured atmaturity. In both cases, endosperms showed a paternal excess phenotype(average maximum endosperm size=800 nuclei, delayed cellularization(+1-2 days relative to 2x-2x crosses wild type) and chalazal endospermhyperplasia) similar to that obtained in 2x-4x crosses between wild typeplants (Table 1).

The mean weight of mature seed collected from pAGL5Met1as plants was 40μg, compared with a mean of 22 μg for 2x-2x seed. This represents anincrease in seed weight compared to the mean of the 2x-2x.

The germination frequency was comparable to that of seed from 2x-2x wildtype crosses—95-100%.

The outcomes of the crosses were variable and depended on the particulartransgenic plant.

The pAGLMet1as gene could be transformed into other crop species such asB. napus and Zea mays, leading to an increase in seed size and seedquality in the transgenic plants. In this case it is most preferable touse MET1 and AGL5 orthologous sequences from B. napus and Zea mays.

b) pAP3Met1as

A proportion of transgenic Arabidopsis plants containing the pAP3Met1asgene were vegetatively normal and produced flowers with the normalcomplement of floral organs.

Arabidopsis containing pAP3Met1as were pollinated with pollen fromwild-type diploid plants or allowed to self pollinate. Endospermdevelopment in the resulting seeds was monitored by confocal microscopy(Scott et al., 1998) and seed weights were measured at maturity. In bothcases, endosperms showed a moderate maternal excess phenotype increasedperipheral endosperm cell number, precocious cellularization andchalazal endosperm hypoplasia qualitatively similar to that obtained in4x-2x crosses between wild type plants (Table 1).

The mean weight of mature seed collected from pAP3Met1as plants is lessthan that of 2x-2x seed.

The germination frequency was comparable to that of seed from 2x-2x wildtype crosses—about 95-100%.

The pAP3Met1as gene could be transformed into other crop species such asB. napus and Zea mays, leading to an decrease in seed size in thetransgenic plants. In this case it is most preferable to use MET1 andAP3 orthologous sequences from B. napus and Z. mays.

Example 5

Promoting Interspecific Hybridization

Tetraploid Arabidopsis thaliana were obtained by the method, known tothose skilled in the art, of Colchicine doubling of a diploid plant.

Cross pollination between tetraploid Arabidopsis thaliana (4x A.thaliana) and Arabidopsis lyrata, results in 100% shriveled seed (FIG.8A) that fail to germinate (Table 3). Abortion is due to endospermfailure resulting from lethal relative genomic imbalance (FIG. 8B). Thispost fertilization hybridization barrier is overcome by introducing theMet1 a/s gene into the 4x A. thaliana parent; the resulting plantsproduce hypomethylated gametes. Cross pollination between a 4x A.thaliana Met1a/s seed plant and Arabidopsis lyrata, results in plumpseed (FIG. 8B) which germinates at high frequency (Table 3). Thisillustrates the utility of hypomethylation, as conditioned by theMet1a/s gene in this example, to promote inter-specific hybridizationbetween two plants that do not normally form viable hybrids.

pAGL5Met1as and pAP3Met1as were transformed into Brassica campestris andBrassica oleraceae via standard methods. Reciprocal crosses between thetransgenic individuals of the two species yield plump seeds whichgerminate to give hybrid plants. Crosses between wild type individualsof the two species result in shriveled seeds which fail to germinate.Hence the two transgenes overcome the normal barrier to interspecifichybridization. The same genes could be used in other species orvarieties to promote hybridization. TABLE 3 Relaxing genomic imprintingthrough hypomethylation can promote or prevent hybrid formation. Outcomeof Cross Endosperm Seed viability Hybrids Cross Phenotype (%germination) formed? 4x A. Thaliana X A. lyrata ME 0 NO 4x A. ThalianaMet1a/s X Moderate PE 95-100 YES A. lyrata 4x A. Thaliana X C. arenosaLethal PE 95-100 YES 2x A. Thaliana X C. arenosa Lethal PE 0 NO 4x A.Thaliana Met1a/s X Lethal PE 0 NO C. arenosaPE, paternal excess as described in Scott et al., 1998.ME, maternal excess as described in Scott et al., 1998.

Example 6

Preventing Interspecific Hybridization

Cross pollination between tetraploid Arabidopsis thaliana (4x A.thaliana) and Cardaminopsis arenosa, results in 100% plump seed (FIG.9A) that germinates at high frequency (Table 3). The hybrid is asynthetic version of a naturally occurring hybrid between these twospecies—Arabidopsis suesica (Chen et al., 1998). Cross pollinationbetween diploid Arabidopsis thaliana (2x A. thaliana) and C. arenosa,results in 100% shriveled seed that fails to germinate (Table 3).Accordingly, C. arenosa can be said to have a genomic strength that issufficiently high to cause seed abortion when combined with 2x A.thaliana, but not when combined with 4x A. thaliana. To demonstrate thathypomethylation can prevent cross hybridization between A. thaliana andC. arenosa the Met1a/s gene was introduced into 4x A. thaliana, and thisplant used as seed parenet in a cross to C. arenosa. Seed from such across is 100% shriveled (FIG. 9B) and fails to germinate (Table 3). Thesame gene could be used in other species or varieties to prevent theproduction of viable hybrid seed.

Example 7

Maternal hypomethylation promotes autonomous endosperm development.

In the absence of fertilization, Arabidopsis plants heterozygous for thefie-1 mutation (fie/FIE) produce seeds with partial endospermdevelopment (Ohad et al., 1996; 1999; see also Table 4 and FIG. 12 A-C).These ‘autonomous’ endosperms consist of a severely reduced number ofendosperm nuclei (compared to wild type controls) and the endospermfails to undergo cellularization. The seed collapses and becomesshriveled at maturity (Table 4). Consequently, the fie mutationconditions only limited endosperm development restricting its utility inthe production of autonomous apomictic seed crops or embryoless seedcrops. Endosperms produced in plants carrying the fis1/mea and fis2mutations are very similar to those of fie/FIE plants, and hence theutility of these genes is also restricted.

Since “fie” endosperms do not contain a paternal genomic contributionone hypothesis is that proper development of the endosperm requires theexpression of paternally derived genes that are subject to maternalimprinting.

When plants heterozygous for the fie mutation are pollinated with wildtype pollen from a 2x wild type plant the ovules carrying the fie alleledevelop into seeds that abort at heart/torpedo stage, while ovulescarrying the wild type FIE allele develop normally (Ohad et al., 1996;1999; Table 4 and FIG. 10). The aborted seeds express a strong paternalcontribution. This suggests that a complex situation with respect toimprinting applies within fertilized and unfertilized fie endosperms.One hypothesis is that the fie mutation lifts imprinting from aproportion of genes normally subject to maternal imprinting: theintroduction of a additional paternal genome following fertilizationgenerates an effective lethal paternal excess such as encountered in a2x-6x wild type cross (Table 1). The failure of fie endosperms todevelopment normally in the absence of fertilization is also accountedfor by this hypothesis, since not all maternally imprinted genes may bederepressed.

Since gametes derived from hypomethylated plants (Met1as and ddm) appearto have no or highly attenuated imprinting, and therefore act in part asgametes of the opposite sex in endosperms, we hypothesized that suchgametes in combination with the fie mutation would promote completeendosperm development. In the first experiment, we used pollen from aMet1as plant [FIE/FIE; MET1 a/s/MET1 a/s] to pollinate a FIE/fieheterzygote [e/FIE; MET1 a/s/MET1 a/s] and found most seeds producedwere plump and viable (Table 4; FIG. 11). The seeds segregate 1:1 forthe FIE/FIE:FIE/fie genotypes, showing that the fie allele istransmissible through the seed parent in this cross. The FIEFIE seedsdisplay a maternal excess phenotype as expected—endospermunder-development (Table 5) and a reduced seed weight (Table 4), whilstthe Fiefie seeds display a moderate paternal excess phenotype (Table 5),similar to that observed in a 2x×4x cross between a wild type A.thaliana plants. When wild type pollen from a diploid plant is used inthis cross, the resulting seeds segregate 1:1 forplump/viable:shriveled/inviable and the ovules containing the fiemutation produce inviable seed since the plump seeds all contain thewild type FIE allele (Table 4; FIG. 10). The abortive seeds display apaternal excess phenotype similar to that observed in a 2x-6x crossbetween wild type A. thaliana plants (FIGS. 3 and 10; Table 5).Therefore, paternal gametes from Met1s plants appear to rescuefiecontaining seeds from lethality by reducing the magnitude of thepaternal excess phenotype. This supports the hypothesis as outlinedabove.

In the second experiment we combined the fie mutation and the Met1asgene into the same individual (see Table 4 and FIG. 12). When theseplants were emasculated and left unpollinated 50% of the ovulesunderwent autonomous endosperm development as expected for ovulescarrying the fie mutation. Confocal microscopy showed that these seedscontain well developed, cellularised endosperms (FIG. 12), with between500-700 peripheral nuclei, a cellularisation time of 5-8 days and avolume of chalazal endosperm between 0.01 and 10× that of a seedproduced in a 2x-2x cross. The mature seeds were shriveled, but weighed15 μg. In contrast, developing ovules of emasculated and unpollinatedFie/fie plants contain very under-developed endosperm that do notcellularize (FIG. 12). These seeds contain about 200 peripheralendosperm nuclei and no recognizable chalazal endosperm. The matureseeds were shriveled and weighed 5 μg. The production of an endospermthat has the main features of a wild type endosperm (numerous peripheralendosperm nuclei, cellularization, and a chalazal endosperm) in plantscontaining both the fie mutation and the Met1as gene shows that thelifting or attenuation of imprinting within the maternal gamete asconditioned by the Met1as gene is sufficient to relieve thedevelopmental block encountered in unpollinated fie ovules. This greatlyextends the utility of the autonomous endosperm mutants (fis1, fis2,fis3, and fie). TABLE 4 Enhancement of endosperm development in fiemutant seeds by hypomethylation. Mature Seed phenotypes Seed weightExtent of (%)¹ Seed viability² (μg)³ endosperm Plump Shrivelled PlumpShrivelled Plum Shrivelled development (%)⁴ seeds seeds seeds seedsseeds seeds Complete Partial FIE/fie × 2x 50 50 95-100 0 25 15 50  50⁵ FIE/fie × 2xmet 100 0 95-100 NA 50% = 15 NA 100 0 50% = 30 FIE/fie 0 100NA 0 NA  5 0  100⁶  emasculate FIE/fie: 0 100 NA 0 NA 20 100 0 2xmetHETemasculateNA, not applicable;FIE/fie, plant heterozygous for the fie mutation;2x, wild type diploid plant;2xmet, plant homozygous for the Met1as gene;FIE/fie, 2xmetHET FIE/fie heterozygous line containing a single Met1asantisense gene (introduced by crossing FIE/fie and Met1as and recoveringappropriate genotype in the F1).¹scored by eye.²determined by germination on soil.³measured as described in Scott et al., 1998.⁴determined by confocal microscopy as described in Scott et al., 1998;complete corresponds to normal development as occurs in control crosses,partial refers to abnormal development such as a failure to cellularizeor develop chalazel endosperm.⁵resembles lethal paternal excess as occurs in 2x-6x crosses 6, asdescribed by Ohad et al., 1999.

TABLE 5 Endosperm development in crosses involving fie, met1a/s and wildtype plants. Fie/FIE X FIE/ Fie/FIE X FIE/ fiemet/met FIE FIE/FIEfie/FIE FIE/FIE fie/FIE Seeds seeds seeds seeds Maximum number of 192637 447 408 P.E. nuclei Timing of endosperm 3-4 DAP 7-8 DAP 5-6 DAP >10DAP cellularization Size of chalazal 0.05-0.1X 3-4X 1X 10-15X Endosperm¹area of maximum cross-section relative to wild type

Example 8

Production of plants that combine the fie mutation and the femalegerm-line specific demethylation gene, AGL5Met1a.

Plants heterozygous for the fie mutation and hemizygous for thepAGL5Met1as gene were generated by making crosses between FIE/fie plantsas pollen parent and plants homozygous for the pAGL5Met1as gene as seedparent. These plants were vegetatively normal and produced normalflowers. When emasculated 50% of the ovules initiated seed developmentwithout fertilization. Confocal microscopy showed that endospermdevelopment was extensive, resulting in a large (500-700 nuclei)cellularized endosperm.

The pAGL5Met1as gene could be introduced into crop species, such as B.napus and Zea mays in which expression of the FIE gene, or any of thegenes that condition autonomous endosperm development, is suppressed orabsent through mutation or the use of transgenic technologies, toproduce promote apomixes or embryoless (pseudoapomictic) seed.Preferably the pAGL5Met1as construct contains B. napus or Z. mays MET1and AGL5 orthologous sequences.

Example 9

The use of FIE down-regulation to paternalise female gametes (polarnuclei)—to increase endosperm size and seed weight.

When plants heterozygous for the fie mutation (Ohad et al., 1996; 1999)are pollinated with pollen from a 2x wild type plant the ovules carryingthe fie allele develop into seeds that abort a heart/torpedo stage,while ovules carrying the wild type FIE allele develop normally (Ohad etal., 1996; 1999; Table 4 and FIG. 10). The aborted seeds express astrong paternal excess phenotype (Table 4; FIG. 10), despite containingonly a single paternal contribution. This suggests that a complexsituation with respect to imprinting applies within fertilized andunfertilized fie endosperms. This is explained by proposing that the fiemutation lifts imprinting from genes normally subject to maternalimprinting (the maternal gametes are thus strongly paternalized): theintroduction of a additional paternal genome following fertilizationgenerates an effective lethal paternal excess (2maternal; 3paternal)such as encountered in a 2x-6x wild type cross (2m:3p) (Table 1).

Since gametes derived from hypomethylated plants (Met1as and ddm) appearto have no or highly attenuating imprinting, and therefore act in partas gametes of the opposite sex in endosperms, such gametes incombination with the fie mutation could promote complete endospermdevelopment. In the first experiment, pollen from a Met1as plant[FIE/FIE; MET1 a/s MET1 a/s] is used to pollinate a FIE/fie heterozygote[fie/FIE; MET1 a/s/MET1 a/s] and most seeds produced were plump andviable (Table 4; FIG. 11). The seeds segregate 1:1 for theFIE/FIE:FIE/fie genotypes, showing that the fie allele is transmissiblethrough the seed parent in this cross. The FIEFIE seeds display amaternal excess phenotype as expected—endosperm under-development (Table5) and a reduced seed weight (Table 4), whilst the Fiefie seeds displaya moderate paternal excess phenotype (Table 5), similar to that observedin a 2x×4x cross between wild type A. thaliana plants. When wild typepollen from a diploid plant is used in this cross, the resulting seedsegregate 1:1 for plump/viable:shriveled/inviable and the ovulescontaining the fie mutation produce inviable seed since the plump seedsall contain the wild type FIE allele (Table 4; FIG. 10). The abortiveseeds display a paternal excess phenotype similar to that observed in a2x-6x cross between wild type A. thaliana plants (FIGS. 3 and 10; Table5). Therefore, paternal gametes from Met1as plants appear to rescuefiecontaining seeds from lethality by reducing the magnitude of thepaternal excess phenotype. As the fie mutation appears to cause strongpaternalization of the maternal gametes (polar nuclei), wild-type FIEmay participate directly in maternal imprinting (as part of theimprinting complex).

The paternalisation of the polar nuclei by the fie mutation is moreextensive than that achieved by met1a/s since a fie×2x cross results inlethal paternal excess (Table 4; FIG. 10), but a met1a/s×2x crossproduces viable paternal excess, with increased endosperm size and seedweight (Table 1). Thus the degree of paternalisation of the polar nucleidetermines the outcome of crosses with pollen from diploid wild typeplants: moderate paternalisation (e.g. Met1a/s) produces a large viableseed due to moderate paternal excess in the endosperm, whereas strongpaternalisation (e.g. fie null mutation) results in seed lethality dueto excessive paternal excess in the endosperm. Modulating FIE expressionmay have application in manipulating endosperm size and seed weight. Thefie mutation used is a null allele (fie-1; Ohad et al., 1999)—nofunctional FIE protein is produced, resulting in strong paternalisationof the polar nuclei, and seed lethality in crosses with wild type pollenfrom a diploid plant. Reducing, but not eliminating the expression ofFIE results in moderate paternalisation of the polar nuclei; the extractlevel of paternalisation being directly related to the amount of FIEprotein expression during female gametogenesis. Reduction in FIEexpression can be achieved using a number of well known methods such asantisense RNA expression against the sense FIE RNA transcript.Incremental reduction in FIE expression, by making use of for exampledifferent, more or less effective, anti-sense lines, identifies a levelof FIE expression that is optimal for producing viable seeds with amaximally increased endosperm size and seed weight.

Suitable anti-sense genes would comprise the FIE promoter drivingtranscription of the anti-sense FIE transcribed region. Other genessuitable to reduce the levels of FIE expression and deliver levels ofpaternalisation of polar nuclei intermediate between a FIE null alleleand the wild type FIE allele include genes encoding fragments of the FIEprotein which recognize and bind to imprinted genes, but are ineffectivein promoting their non-expression in the endosperm (e.g. because therepressive complex cannot form or cannot be maintained).

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1. A method for the production of seeds, comprising the step ofpermitting pollination of a plant transformed with an Arabidopsis ddm1nucleic acid sequence effective for reducing levels of cytosinemethylation in repetitive sequences, said nucleic acid sequence operablylinked to a promoter that targets expression to female germ line cells,wherein seeds that develop on said plant have increased mean seed weightcompared to the mean seed weight of seeds that develop on acorresponding pollinated plant that lacks said nucleic acid sequence. 2.The method of claim 1, wherein said nucleic acid sequence comprises anArabidopsis ddm1 antisense nucleic acid.
 3. The method of claim 1,wherein said nucleic acid sequence comprises a full or partial sensecopy of an Arabidopsis ddm1 nucleic acid sequence.
 4. The method ofclaim 3, wherein said sense copy is a partial sense copy.
 5. The methodof claim 1, wherein said promoter targets expression to female gameticcells.
 6. A plant transformed with a nucleic acid comprising a promoterthat targets expression to female germ line cells operatively linked toan Arabidopsis ddm1 nucleic acid sequence effective for reducing levelsof cytosine methylation in repetitive sequences.